NGS Sequencing

Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.

Targeted Testing

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

Analytical sensitivity by DNA sequencing will be limited because a significant number of gross deletions of the MTM1 gene are reported. Clinical sensitivity is problematic to predict due to genetic heterogeneity of this disorder. From a cohort of 60 patients, seven were found to have MTM1 mutations (Laporte et al. Nature Genet 13:175-182, 1996).

Pricing Comment

Turnaround Time

Clinical Features

X-linked centronuclear myopathy (CNMX; OMIM 310400), also known as X-linked myotubular myopathy-1 (MTM1), is a severe congenital myopathy in affected males and is caused by mutations in the MTM1 gene (Laporte et al. Nature Genet 13:175-182, 1996). Pregnancies involving males with CNMX are complicated by polyhydramnios secondary to decreased fetal swallowing. Decreased fetal movement is also noted, and some newborns are found to have congenital eventration of the diaphragm leading to respiratory failure (Heckmatt et al. Brain 108:941-964, 1985; Moerman et al. Am J Med Genet 27:213-218, 1987). Newborns are extremely hypotonic and require ventilator support to avoid hypoxia. Facial, extraocular, and neck muscles are always affected. Other features seen at birth include body length greater than the 90th percentile, large head circumference, elongated face, and long fingers and toes (Joseph et al. Am J Med Genet 59:168-173, 1995). A review of medical records of 55 male CNMX patients showed that survivability beyond 1 year of age was common (Herman et al. J Pediat 134:206-214, 1999). Most surviving patients (80%) in the study were ventilator-dependent and their muscle symptoms were non-progressive. Other complications observed in the surviving patients included liver dysfunction, rapid growth with advanced bone age, and pyloric stenosis. Muscle biopsies in CNMX reveal centrally placed nuclei, and most patients have reduced myotubilarin protein in a variety of cell types (Laporte et al. Ann Neurol 50:42-46, 2001). Female carriers are usually asymptomatic, however, cases of obligate carriers have been reported with a range of clinical symptoms. Symptoms in mildly affected female carriers include facial weakness and limb-girdle weakness (Wallgren-Pettersson et al. J Med Genet 32:673-679, 1995). Childhood-onset cases generally have skewed X inactivation and manifestations of moderate facial and neck flexor weakness, scapular winging, weakness and wasting of proximal arm muscles, elevated hemidiaphragm, and generalized, slowly progressive weakness in adulthood (Tanner et al. Hum Genet 104:249-253, 1999; Grogan et al. Neurology 64:1638-1640, 2005). In one case of a carrier female with limb-girdle and facial weakness, skewed X inactivation was not observed (Sutton et al. Neurology 57:900-902, 2001). Clinical features of a female with congenital onset of disease include fetal hypokinesia and severe hypotonia at birth. The affected infant also had absent deep tendon reflexes, a weak cry, ptosis, and a high-arched palate (Schara et al. Neurology 60:1363-1365, 2003). The patient later developed limb girdle and facial weakness.

Genetics

Centronuclear myopathy is a genetically heterogeneous disorder. One autosomal recessive form and several autosomal dominant forms are known. MTM1-related centronuclear myopathy is inherited as an X-linked recessive disorder. Over 250 different mutations of the MTM1 gene are reported. The largest class of mutations is substitution of amino acids followed by small deletions, splice site mutations, nonsense mutations, small insertions, and gross deletions (Laporte et al. Hum Mutat 15:393-409, 2000; Zanoteli et al. Am J Med Genet 134A:338-340, 2005).

Testing Strategy

For this NextGen test, the full coding regions plus ~10 bp of non-coding DNA flanking each exon are sequenced for the gene listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

Indications for Test

Male patients with clinical features of centronuclear myopathy and with centrally placed nuclei in muscle. Female patients with clinical features consistent with those previously reported in obligate carriers.

TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are covered by Sanger sequencing. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood). Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Ship blood tubes at room temperature in an insulated container. Do not freeze blood.

During hot weather, include a frozen ice pack in the shipping container.
Place a paper towel or other thin material between the ice pack and the blood tube.

In cold weather, include an unfrozen ice pack in the shipping container as insulation.

At room temperature, blood specimen is stable for up to 48 hours.

If refrigerated, blood specimen is stable for up to one week.

Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.

For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.

DNA may be shipped at room temperature.

Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.

We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

PreventionGenetics should be notified in advance of arrival of a cell culture.

Culture and send at least two T25 flasks of confluent cells.

Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.

Send specimens in insulated, shatterproof container overnight.

Cell cultures may be shipped at room temperature or refrigerated.

Label the flasks with the patient name, date of birth, and/or ID number.

We strongly recommend maintaining a local back-up culture. We do not culture cells.